The most successful uses of in vivo EPR have been non-invasive measurement of oxygen, nitric oxide, bioradicals, pH and redox state, with applications in oncology, cardiology, neuroscience and toxicology. These studies have been performed only in small size subjects due to fundamental limitations associated with the traditional high-frequency detection scheme. Current generation EPR systems typically use GHz or higher frequencies (L-, X-, Q- band) to achieve the required resolution. There are several key factors, which make in vivo EPR methods of human body at high frequencies extremely difficult, the principal ones being limits on the size and shape of the systems that can be measured and safety issues related to the absorption of high-level RF by biological systems. We will develop a new ultra-low field in vivo EPR spectrometer system that is safe for use in humans and has high sensitivity and good penetration depth. The new device will overcome the limitations associated with conventional EPR at high frequencies yet still achieve 100 to 1000 fold improved SNR over existing technology. We will achieve this goal by employing two state- of-the-art technologies developed in our lab: (1) a new microwave superconducting quantum interference device (MSQUID) (2) a cryogen-free, superconducting gradiometer system. Operation at 5MHz with a low power RF excitation will completely eliminate the problems associated with finite penetration depth and sample-heating. Our aims are: 1A) Demonstrate ?M-level EPR signal detection (both CW and pulse methods) using paramagnetic samples (e.g. spin probes) at room temperature using cryogenic detection with the new hybrid SQUID readout system. 1B) Demonstrate the NMR signal detection using the same detection system, with hybrid NMR/EPR spectroscopy systems in mind. 2) Use a cryogen-free cooling system with a large field-of-view detection coil to demonstrate EPR spectroscopy performance, using parameters suitable for the human study. 3) Demonstrate detection and measurement of electron paramagnetic resonance in biological systems at physiologically relevant concentrations. 3A. Using living suspensions of Chlorella pyroidenosa, we will attempt to detect the expected electron spin resonance under dark conditions, and to detect and quantify the ESR increase that occurs during photosynthesis. These technical aims form the foundations for the application of the advanced technology to human use. With the long term goal of a clinical device, we will also perform limited human testing. Our aim3B is to perform in vivo human experiments to detect increased free radical concentration during intense exercise. Our ultimate objective is to create a device for human in vivo biomedical EPR that is practical from the perspectives of safety, cost, siting and complexity, without compromising sensitivity and signal quality. PUBLIC HEALTH RELEVANCE (provided by applicant): Free radicals play a major role in human diseases such as iron sulfate poisoning, ionizing radiation, oxygen toxicity in premature infants treated with hyperbaric oxygen, ultraviolet radiation-induced cancer and very likely in Parkinson's, Alzheimer's and other neurodegenerative disorders. Electron paramagnetic resonance (EPR) is a non-invasive spectroscopic technique to detect and measure free radicals in chemical and biological system that has been applied in- vivo EPR to measure oxygen, nitric oxide, bioradicals, pH and redox state, with applications in oncology, cardiology, neurology and toxicology. To date, these studies have been performed only in small size subjects due to fundamental technical and safety limits of the conventional high-frequency detection scheme. We propose here to develop a novel ultra low-frequency EPR approach using a state-of-the-art magnetic flux sensor to enable safe, cost- effective and practical in vivo EPR humans, with an instrument ultimately capable of simultaneous NMR measurement.